Diversity of laccase among Cryptococcus neoformans serotypes

The pathogenic yeast C. neoformans is classified into three varieties with five serotypes; var. grubii (serotype A), var. neoformans (serotype D), var. gattii (serotypes B and C), and serotype AD. Melanin is a virulence factor in the species, and its biosynthesis is catalyzed by laccase, encoded by the LAC1 gene. In order to estimate the natural variability of the LAC1 gene among Cryptococcus serotypes, the laccase protein sequence from 55 strains was determined and the phylogenetic relationships between cryptococcal and related fungal laccases revealed. The deduced laccase proteins consisted of 624 amino acid residues in serotypes A, D and AD, and 613 to 615 residues in serotypes B and C. Intra-serotype amino acid variation was marginal within serotypes A and D, and none was found within serotypes AD and C. Maximum amino acid replacement occurred in two serotype B strains. The similarity in the deduced sequence ranged from 80 to 96% between serotypes. The sequence in the copper-binding regions was strongly conserved in the five serotypes. The laccases of the five serotypes were grouped together in the same clade of the phylogenetic tree reconstructed from different fungal laccases, suggesting a monophyletic clade.     

Requirement of a Tsp2-type tetraspanin for laccase repression and stress resistance in the basidiomycete Cryptococcus neoformans

Fungal laccases have been widely used in industry. The expression of laccase often is repressible by the primary carbon source glucose in many fungi. The underlying basis is largely unclear. We demonstrate here that a gene, TSP2-1, was required for laccase repression by glucose in the basidiomycete Cryptococcus neoformans. TSP2-1 encodes a Tsp2-type tetraspanin. The disruption of TSP2-1 resulted in constant melanin formation and the expression of the laccase gene LAC1. This derepression phenotype was restorable by 10 mM exogenous cyclic AMP (cAMP). A capsule defect in the mutant tsp2-1Δ also was restored by cAMP. The results indicate an interaction of Tsp2-1 with the cAMP-dependent protein kinase A (PKA) pathway that has been shown to modulate laccase repression and capsule biosynthesis in this fungus. Other roles of TSP2-1, e.g., in maintaining cell membrane integrity and stress resistance, also were defined. This work reveals a Tsp2-1-dependent glucose repression in C. neoformans. The function of Tsp2-type tetraspanin Tsp2-1 is described for the first time.     

Cryptococcus neoformans virulence gene discovery through insertional mutagenesis

Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.     

Enzymes that counteract nitrosative stress promote fungal virulence

Enzymes that protect cells from reactive oxygen species (superoxide dismutase, catalase, peroxidase) have well-established roles in mammalian biology and microbial pathogenesis. Two recently identified enzymes detoxify nitric oxide (NO)-related molecules; flavohemoglobin denitrosylase consumes NO, and S-nitrosoglutathione (GSNO) reductase metabolizes GSNO. Although both enzymes protect microorganisms from nitrosative challenge in vitro, their relevance has not been established in physiological contexts. Here we studied their biological functions in Cryptococcus neoformans, an established human fungal pathogen that replicates in macrophages and whose growth in vitro and in infected animals is controlled by NO bioactivity. We show that both flavohemoglobin denitrosylase and GSNO reductase contribute to C. neoformans pathogenesis. FHB1 and GNO1 mutations abolished NO- and GSNO-consuming activity, respectively. Growth of fhb1 mutant cells was inhibited by nitrosative challenge, whereas that of gno1 mutants was not. fhb1 mutants showed attenuated virulence in a murine model, and virulence was restored in iNOS(-/-) animals. Survival of the fhb1 mutant was also reduced in activated macrophages and restored to wild-type by inhibition of NOS activity. Combining mutations in flavohemoglobin and GSNO reductase, or flavohemoglobin and superoxide dismutase, further attenuated virulence. These studies illustrate that fungal pathogens elaborate enzymatic defenses against nitrosative stress mounted by the host.     

The role and mechanism of diacylglycerol-protein kinase C1 signaling in melanogenesis by Cryptococcus neoformans

The fungus Cryptococcus neoformans is an opportunistic human pathogen that causes a life-threatening meningoencephalitis by expression of virulence factors such as melanin, a black pigment produced by the cell wall-associated enzyme laccase. In previous studies (Heung, L. J., Luberto, C., Plowden, A., Hannun, Y. A., and Del Poeta, M. (2004) J. Biol. Chem. 279, 21144-21153) we proposed that the sphingolipid enzyme inositol-phosphoryl ceramide synthase 1 (Ipc1) regulates melanin production through the generation of diacylglycerol (DAG), which was found to activate in vitro protein kinase C1 (Pkc1). Here, we investigated the molecular mechanisms by which DAG regulates Pkc1 in vivo and the effect of this regulation on laccase activity and melanin synthesis. To this end we deleted the putative DAG binding C1 domain of C. neoformans Pkc1 and found that the C1 deletion abolished the activation of Pkc1 by DAG. Deletion of the C1 domain repressed laccase activity and, consequently, melanin production. Finally, we show that these biological effects observed in the C1 deletion mutant are mediated by alteration of cell wall integrity and displacement of laccase from the cell wall. These studies define novel molecular mechanisms addressing Pkc1-laccase regulation by the sphingolipid pathway of C. neoformans, with important implications for understanding and targeting the Ipc1-Pkc1-laccase cascade as a regulator of virulence of this important human pathogen.     

Immune Modulation Mediated by Cryptococcal Laccase Promotes Pulmonary Growth and Brain Dissemination of Virulent Cryptococcus neoformans in Mice

C. neoformans is a leading cause of fatal mycosis linked to CNS dissemination. Laccase, encoded by the LAC1 gene, is an important virulence factor implicated in brain dissemination yet little is known about the mechanism(s) accounting for this observation. Here, we investigated whether the presence or absence of laccase altered the local immune response in the lungs by comparing infections with the highly virulent strain, H99 (which expresses laccase) and mutant strain of H99 deficient in laccase (lac1Δ) in a mouse model of pulmonary infection. We found that LAC1 gene deletion decreased the pulmonary fungal burden and abolished CNS dissemination at weeks 2 and 3. Furthermore, LAC1 deletion lead to: 1) diminished pulmonary eosinophilia; 2) increased accumulation of CD4+ and CD8+ T cells; 3) increased Th1 and Th17 cytokines yet decreased Th2 cytokines; and 4) lung macrophage shifting of the lung macrophage phenotype from M2- towards M1-type activation. Next, we used adoptively transferred CD4+ T cells isolated from pulmonary lymph nodes of mice infected with either lac1Δ or H99 to evaluate the role of laccase-induced immunomodulation on CNS dissemination. We found that in comparison to PBS treated mice, adoptively transferred CD4+ T cells isolated from lac1Δ-infected mice decreased CNS dissemination, while those isolated from H99-infected mice increased CNS dissemination. Collectively, our findings reveal that immune modulation away from Th1/Th17 responses and towards Th2 responses represents a novel mechanism through which laccase can contribute to cryptococcal virulence. Furthermore, our data support the hypothesis that laccase-induced changes in polarization of CD4+ T cells contribute to CNS dissemination.     

新型隐球酵母氯离子通道在阳离子内稳态中的趋异作用

新型隐球酵母Cryptococcus neoformans有两个变种（varieties）,即grubii和neoformans。目前研究最多的两个菌株H99（血清型A）和JEC21（血清型D）分别代表这两个变种,两者的毒性差别显著,为研究新型隐球酵母菌株间毒性的进化提供了良好模型。我们通过比较JEC21的clc1-突变体Tx1与早先鉴定的H99 clc1-菌株Mlac3发现,JEC21 CLC1同样决定铜离子的吸收。Tx1中丧失的漆酶活力可以通过外源Cu2+的加入得以恢复,而漆酶基因LAC1的转录与野生